Figures and data
Lamininϒ1 and collagen IV form 3D cages of ECM for megakaryocytes, directly connected to the sinusoidal basement membrane
(A) Left panel. Schematic representation of the experimental workflow for 2D imaging of immunostained bone marrow cryosections from WT mice. Confocal imaging is performed on single ultrathin sections with an axial resolution of 250 µm. Right panel. Representative 2D images of a sinusoid-associated megakaryocyte immunostained for laminin ϒ1 (red), GPIbβ (white) and FABP4 (cyan). Cell nuclei are visualized with DAPI (blue) (from one out of three independent IF experiments).
(B) Left panel. Schematic representation of the experimental workflow for 3D analysis of whole-mount bone marrow preparations from WT mice. A stack of confocal images covering half the depth of the megakaryocyte is acquired and then z-projected to create a maximum projection image. Right panel. Representative 3D images of sinusoid-associated megakaryocyte immunostained for laminin ϒ1 (red), GPIbβ (white) and FABP4 (cyan) (from one out of three independent IF experiments).
(C) Representative 3D images of sinusoid-associated megakaryocyte immunostained for collagen IV (green) and GPIbβ (white).
(D) Bone marrow-isolated megakaryocyte maintaining an ECM cage. Left panel. Schematic of the experimental procedure used to isolate mouse bone marrow megakaryocytes. Right panel. Maximal projection 3D images showing the persistence of the ECM cages (collagen IV in green) around freshly isolated megakaryocytes (GPIbβ in white).
*, sinusoid lumen; arrowheads, basement membrane-cage connection; BM, bone marrow; bm, basement membrane; MK, megakaryocyte; Bars, 10 µm.
Reduced laminin cage and megakaryocyte-sinusoid interactions in Lama4−/− mouse bone marrow.
(A-B) Depletion of laminin α4 leads to a reduction in the laminin ϒ1 deposition, but in collagen IV, in the cage around megakaryocytes and in the sinusoid basement membrane. Left panels. Representative maximal projections showing the immunostaining of laminin γ1 (in red) or collagen IV (in green) in the Lama4−/− compared to WT mice. Two magnifications are shown for Lama4−/− mouse. Right panels. Quantification of laminin γ1 and collagen IV surface coverage per megakaryocyte and per basement membrane surface (laminin: 6<cage<17 and 15<bm<17; collagen IV: 6<cage<16 and 11<bm<21 expressed as a percentage, ****P>0.001 unpaired t-Test).
(C) Depletion of laminin α4 leads to a decrease in the sinusoid-associated megakaryocytes. Left panels. Representative maximal projections showing the immunostaining of laminin γ1 (in red) or megakaryocytes (GPIbβ in white) in the bone marrow of Lama4−/− and WT mice. Right panels. Quantification of the total number of megakaryocytes per surface unit (s.u., 12,945 μm2, n=3 for each genotype) and of sinusoid-associated megakaryocytes (n=3, ****P>0.001 unpaired t-Test) in WT (grey) and Lama4−/− (dark) mice. Arrows point to megakaryocytes which are not associated with sinusoids (pMK).
bm, basement membrane; MK, megakaryocyte; pMK, MK in the parenchyma; Bar, 10 µm.
Integrin-mediated control of the 3D ECM cage around megakaryocytes
(A) Representative 2D images of Pf4cre bone marrow cryosections (250 nm) showing a sinusoid-associated megakaryocyte immunostained for β1 integrin (MAB1997 in yellow). Right: the boxed area is shown at a higher magnification.
(B) Activated β1 integrins form functional adhesion structures around megakaryocyte surfaces. Representative 2D images of Pf4cre bone marrow cryosections showing a sinusoid-associated megakaryocyte immunostained for activated β1 integrin (9EG7 in cyan), for GPIbβ (white) and laminin (red, upper panels) or collagen IV (green, lower panels). Right: Magnification of the boxed area showing co-localization of laminin and 9EG7.
(C) Depletion of β1 and β3 integrins leads to a reduction in the laminin deposition on the surface of megakaryocytes. Upper panels. Representative 3D images showing a decrease in laminin deposition (red) on Itgb1−/−/Itgb3−/− megakaryocytes compared to Pf4cre. Top panels. (Left) Quantification of laminin surface coverage per megakaryocyte (in %, 17<n<19 as indicated in the bars, ***P>0.001 unpaired t-Test), (Middle) expression profile of the laminin staining along straight-lines (25 µm long) visible as white lines in the confocal images, and (Right) quantification of mesh sizes (in µm, 14<n<16, **P<0.01 Mann-Whitney).
*, sinusoid lumen; MFI, mean fluorescence intensity; MK, megakaryocyte; pm, plasma membrane; Bars, 10 µm.
Integrins protects megakaryocytes from entering the bloodstream as whole cells
(A) Higher proportion of intravasation events in mice lacking β1/β3 integrins. Upper panel. Representative confocal images of Pf4cre and Itgb1−/−/Itgb3−/−whole-mount bone marrow immunostained for GPIbβ (white) and FABP4 (cyan). Top panel. Quantification of megakaryocyte intravasation and circulating megakaryocytes (3 mice minimum for each genotype, 213<n<397 for Pf4cre and Itgb1−/−/Itgb3−/−, ** P<0.001 Mann Whitney).
(B) Left panel. Quantification of the laminin γ1 deposition in the ECM cage in single knockout integrins and in GPVI knockout. Right panel. Quantification of the intravasation events in single knock-out mice showing that both integrins are essential for the proper anchoring of megakaryocytes in their vascular niche.
(C) Intravital two-photon imaging of Itgb1−/−/Itgb3−/−mouse calvarial bone marrow stained with intravenously injected AF488-conjugated anti-GPIX antibody and rhodamin dextran. The white arrow indicates an intrasinusoidal Itgb1−/−/Itgb3−/− megakaryocyte, dotted lines illustrate the sinusoid wall and the values in the left corner show the time-lapses. Quantification of circulating megakaryocytes, expressed as a percentage of the total number of megakaryocyte (from 3 independent experiments, 130<n<136 as indicated in the legend, P<0.05, Mann-Whitney).
(D) Increased number of pyrenocytes (arrow, GPIbβ green, DAPI nucleus) within the pulmonary microvessels of Itgb1−/−/Itgb3−/− mice. Cyan dotted lines indicate the vessel wall. Quantification of the intravascular pyrenocytes (from 5 independent experiments, 28<n<149 as indicated in bars, **P<0.01, Mann-Whitney).
(E) Two TEM image showing intravascular entire Itgb1−/−/Itgb3−/− megakaryocyte.
*, sinusoid wall; FG, fibrinogen; FN, fibronectin; FG, fibrinogen; n, nucleus; sMK, sinusoid-associated MK; pMK, MK in the parenchyma; PPT, proplatelets; Bars in A-G, 10 μm; Bar in E, 5 µm; Bar in G, 30µm.
Integrins promote megakaryocyte adhesion to the ECM components of the bone marrow
(A) Impaired adhesion and spreading of Itgb1−/−/Itgb3−/− MK. Representative SEM images depicting bone marrow-derived megakaryocytes adhering on laminin. Spreading (hatched bars) and round (grey bars) megakaryocytes were counted following 3h incubation on laminin, fibronectin (FN), and fibrinogen (FG) (in %) (from 4-6 independent experiments).
(B) Microfluidic flow chamber to study megakaryocyte adhesion efficiency. Representatives bright field images showing that upon flow application, Itgb1−/−/Itgb3−/−megakaryocytes detach from fibrillary fibronectin protein, while Pf4Cre MKs remain attached. Quantification of the detachment of Pf4cre and Itgb1−/−/Itgb3−/− megakaryocytes on laminin, fibrillar fibronectin and fibrinogen (from 5 to 7 independent experiments,**P<0.01, ***P<0,001, Mann-Whitney).
(C) Reduced physical anchoring of Itgb1−/−/Itgb3−/− megakaryocytes to BM. Representatives bright field images of the ten femur BM sections placed in an incubation chamber (left panel), of the box (center panel) and of the megakaryocytes released from the periphery of the explants (right panel). Quantification of the number of Pf4cre and Itgb1−/−/Itgb3−/− megakaryocytes released from the explants following 3h (from 10 to 13 independent experiments, 594<n<1095 for Pf4cre and Itgb1−/−/Itgb3−/−, *P<0.05, unpaired t-test).
dotted lines, MK detachment; MK, megakaryocytes, FN, fibronectin; FG, fibrinogen; MK, megakaryocytes, n, number of cells studied; Bars in A, 10 μm; Bars in B, 20 µm; Bars in B, 30 µm.
Maturation of the 3D ECM cage is correlated with maturation of the DMS in megakaryocytes
(A) MMP inhibition leads to a densification of the 3D ECM cage. Upper panels. Representative 3D confocal images showing a significant increase in collagen IV deposition (green) on the megakaryocyte surface in treated mice treated with the intravenous cocktail of protease inhibitors (B + I). Top panels. (Left) Quantification of collagen IV fluorescence showed a shortening of collagen IV fibers in treated mice compared to that in control mice (from 3-5 independent experiments, 20<n<22 as indicated in the bars, ***P<0;001, Mann-Whitney), (Middle) Histograms of fluorescence intensity versus distance showed an increase in cross-linking with a reduction in pore size (white lines of 25µm length are visible in the confocal images), (Right) Reduction in mesh sizes in treated mice (from 3 independent experiments, 7<n<12, ***P>0.001, t-test).
(B) MMP inhibition affects megakaryocyte growth (indicated by arrows). Representative confocal images from DMSO vs B+I treated mice immunostained for GPIbβ (white) and FABP4 (cyan), zoomed-in images showing the difference in megakaryocyte size between the two groups. Quantification of the number of megakaryocytes per bone marrow area (194 x 194 µm) (from 3 independent experiments, 229<n<483 as indicated in the bars, ***P<0.0001, Mann-Whitney).
(C) TEM observation revealed the presence of numerous immature megakaryocytes (stage II) in treated mice as compared to fully mature megakaryocytes in control mice (stage III). (Middle) Quantification of the total number of megakaryocyte s (145<n<169 as indicated in bars, **P<0.01, Mann-Whitney) and in the proportion of immature megakaryocytes (stage II) in the B + I group (from 3 independent experiments, *P < 0.05, **P < 0.01, one way ANOVA Dunn’s).
(D) Representatives bright field images of the megakaryocytes (arrows) released from the periphery of the control and treated explants. Quantification of the number of megakaryocytes released following 3h and 6h (from 6 independent experiments, 370<n<783 for DMSO and B+I, *P<0.05, unpaired t-test).
B + I, batimastat + Ilomastat; sMK, sinusoid-associated MK; pMK, MK in the parenchyma; n, number of cells studied; Bars in A and B, 10 μm; Bars in C, 5 µm; Bar in D, 50 µm.
